Project Summary: Type 1 diabetes (T1D) results from a breakdown in immune tolerance that progresses to the destruction of the insulin producing beta cells of the pancreatic islets. A large number of genetic and environmental factors contribute to the incidence of disease. One of the strongest non-MHC genetic risk factors is a single nucleotide polymorphism, C1858T, in the gene that encodes the tyrosine phosphatase non-receptor type 22, PTPN22, a protein expressed in bone marrow derived cells. It is not known how PTPNR620W enhances T1D, or the many other autoimmune diseases associated with this allele. In the absence of other pro-autoimmune loci, the mouse ortholog, PTPN22R619W does not lead to autoimmunity. We have used Crispr/Cas genome editing to introduce the pro-autoimmune allele of PTPN22 into the non-obese diabetes (NOD) mouse that spontaneously develops T1D. NOD mice harboring this mutation demonstrate accelerated production of anti-insulin antibodies and T1D, the same phenotype exhibited by humans expressing PTPN22R620W. This proposal will use this murine line to reveal the mechanism of enhanced disease by pursuing the following specific aims: Specific Aim 1: The basis for accelerated loss of CD4 T cell tolerance in R619W NOD mice. Individual CD4 cells from wildtype and mutant NOD mice specific for islet antigens will be compared by flow cytometry and single-cell gene expression to reveal phenotypic and functional differences that may explain enhanced pathogenesis of T cells from mutant mice. Specific Aim 2. Tolerance and homeostasis of B lymphocytes in R619W mice. We will compare the frequency and functionality of insulin specific B cells, from R619W and NOD, at different developmental stages, and following activation. Specific Aim 3. The relative contribution of R619W expressing CD4 T and B cells to antibody production and development of disease will be assessed. The presence of anti-islet antibodies is a predictive marker for development of T1D; however, the cellular and mechanistic basis for this association is not known. As both are accelerated by R619W, we have a unique opportunity to determine whether the expression of the mutation in the same or different cell type, is the basis for enhanced autoantibodies and enhanced disease. This may be of significance in selecting among different forms of immune therapy for patients.